Background:Antimicrobial resistance (AMR) is now considered one of the greatest global health threats. This is further compounded by a lack of new antibiotics in development. Antimicrobial stewardship programmes can improve and optimize the use of antibiotics, thereby increasing the cure rates of antibiotic treatment and decreasing the problem of AMR. In addition, diagnostic and antimicrobial stewardships in the pathology laboratories are useful tools to guide clinicians on patient treatment and to stop the inappropriate use of antibiotics in empirical treatment or narrow antibiotics. Medical Laboratory Scientists are at the forefront of performing antibiotics susceptibility testing in pathology laboratories, thereby helping clinicians to select the appropriate antibiotics for patients suffering from bacterial infections.Methods:This cross-sectional study surveyed personal antimicrobial usage, the knowledge and awareness on AMR, and antimicrobial stewardship, as well as barriers to antimicrobial susceptibility testing among medical laboratory scientists in Nigeria using pre-tested and validated questionnaires administered online. The raw data were summarized and exported in Microsoft Excel and further analyzed using IBM SPSS version 26.Results:Most of the respondents were males (72%) and 25–35 years old (60%). In addition, the BMLS degree was the highest education qualification most of the respondents (70%) achieved. Of the 59.2% of the respondents involved in antibiotics susceptibility testing, the disc diffusion method was the most commonly used (67.2%), followed by PCR/Genome-based detection (5.2%). Only a small percentage of respondents used the E-test (3.4%). The high cost of testing, inadequate laboratory infrastructure, and a lack of skilled personnel are the major barriers to performing antibiotics susceptibility testing. A higher proportion of a good AMR knowledge level was observed in male respondents (75%) than females (42.9%). The knowledge level was associated with the respondent’s gender (p= 0.048), while respondents with a master’s degree were more likely to possess a good knowledge level of AMR (OR: 1.69; 95% CI: 0.33, 8.61).Conclusion:The findings of this study indicate that Nigerian medical laboratory scientists had moderate awareness of AMR and antibiotic stewardship. It is necessary to increase investments in laboratory infrastructure and manpower training, as well as set up an antimicrobial stewardship programme to ensure widespread antibiotics susceptibility testing in hospitals, thereby decreasing empirical treatment and the misuse of antibiotics.