Non-steroidal anti-inflammatory drugs (NSAIDs) are increasingly recognized as potential environmental contaminants that may induce toxicity in aquatic ecosystems. This 3-week microcosm experiment explores the acute impacts of NSAIDs, including diclofenac (DCF), ibuprofen (IBU), and acetylsalicylic acid (ASA), on bacterial communities using a wide range of these substances (200–6000 ppm). The results showed that the NSAID-treated microcosms had higher cell count values than control samples, though the diversity of microbial communities decreased. The isolated heterotrophic bacteria mostly belonged toProteobacteria, particularlyKlebsiella. Next-generation sequencing (NGS) revealed that NSAIDs altered the structure of the bacterial community composition, with the proportion ofProteobacteriaaligning with the selective cultivation results. Bacteria had higher resistance to IBU/ASA than to DCF. In DCF-treated microcosms, there has been a high reduction of the number ofBacteroidetes, whereas in the microcosms treated with IBU/ASA, they have remained abundant. The numbers ofPatescibacteriaandActinobacteriahave decreased across all NSAID-treated microcosms.VerrucomicrobiaandPlanctomyceteshave tolerated all NSAIDs, even DCF.Cyanobacteriahave also demonstrated tolerance to IBU/ASA treatment in the microcosms. The archaeal community structure was also impacted by the NSAID treatments, withThaumarchaeotaabundant in all microcosms, especially DCF-treated microcosms, whileNanoarchaeotais more typical of IBU/ASA-treated microcosms with lower NSAID concentrations. These results indicate that the presence of NSAIDs in aquatic environments could lead to changes in the composition of microbial communities.