BackgroundPolymerase chain reaction (PCR) testing for COVID-19 was crucial in Australia’s prevention strategy in the first 2 years of the pandemic, including required testing for symptoms, contact with cases, travel, and certain professions. However, several months into the pandemic, half of Australians were still not getting tested for respiratory symptoms, and little was known about the drivers of and barriers to COVID-19 PCR testing as a novel behavior at that time.ObjectiveWe aimed to identify and address COVID-19 testing barriers, and test the effectiveness of multiple eHealth interventions on knowledge for people with varying health literacy levels.MethodsThe intervention was developed in 4 phases. Phase 1 was a national survey conducted in June 2020 (n=1369), in which testing barriers were coded using the capability-opportunity-motivation-behavior framework. Phase 2 was a national survey conducted in November 2020 (n=2034) to estimate the prevalence of testing barriers and health literacy disparities. Phase 3 was a randomized experiment testing health literacy–sensitive written information for a wide range of barriers between February and March 2021 (n=1314), in which participants chose their top 3 barriers to testing to view a tailored intervention. Phase 4 was a randomized experiment testing 2 audio-visual interventions addressing common testing barriers for people with lower health literacy in November 2021, targeting young adults as a key group endorsing misinformation (n=1527).ResultsIn phase 1, barriers were identified in all 3 categories: capability (eg, understanding which symptoms to test for), opportunity (eg, not being able to access a PCR test), and motivation (eg, not believing the symptoms are those of COVID-19). Phase 2 identified knowledge gaps for people with lower versus higher health literacy. Phase 3 found no differences between the intervention (health literacy–sensitive text for top 3 barriers) and control groups. Phase 4 showed that a fact-based animation or a TikTok-style video presenting the same facts in a humorous style increased knowledge about COVID-19 testing compared with government information. However, no differences were found for COVID-19 testing intentions.ConclusionsThis study identified a wide range of barriers to a novel testing behavior, PCR testing for COVID-19. These barriers were prevalent even in a health system where COVID-19 testing was free and widely available. We showed that key capability barriers, such as knowledge gaps, can be improved with simple videos targeting people with lower health literacy. Additional behavior change strategies are required to address motivational issues to support testing uptake. Future research will explore health literacy strategies in the current context of self-administered rapid antigen tests. The findings may inform planning for future COVID-19 variant outbreaks and new public health emergencies where novel testing behaviors are required.Trial RegistrationAustralian New Zealand Clinical Trials Registry ACTRN12621000876897, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=382318 ; Australian New Zealand Clinical Trials Registry ACTRN12620001355965, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=380916&isReview=true