Background Mass testing campaigns were proposed in France during the first wave of the COVID-19 pandemic to detect and isolate asymptomatic individuals infected by SARS-CoV-2. During mass testing in Saint-Étienne (February 2021), we performed a survey of the general population. Objective We evaluated, on the scale of a city’s population, the literacy level about SARS-CoV-2 transmission, barrier gesture respect, and isolation acceptability or possibility in case of SARS-CoV-2 infection. Methods We used the validated CovQuest-CC questionnaire. Data were analyzed and correlated with volunteer characteristics and their SARS-CoV-2 screening results using multivariate analysis. Results In total, 4707 participants completed the CovQuest-CC questionnaire. Multivariate analysis revealed that female sex was a determinant of a higher score of knowledge about SARS-CoV-2 transmission (adjusted β coefficient=0.14, 95% CI 0.04-0.23; corrected P=.02). Older ages of 50-59 years (adjusted β coefficient=0.25, 95% CI 0.19-0.31; corrected P<.001) and ≥60 years (adjusted β coefficient=0.25, 95% CI 0.15-0.34; corrected P<.001) were determinants of a higher score on barrier gesture respect compared to ages 20-49 years considered as reference. Female sex was also a determinant of a higher score on barrier gesture respect (adjusted β coefficient=0.10, 95% CI 0.02-4.63; corrected P<.001). The knowledge score was correlated with the score on barrier gesture respect measures (adjusted β coefficient=0.03, 95% CI 0.001-0.004; corrected P=.001). Older ages of 50-59 years (adjusted β coefficient=0.21, 95% CI 0.13-0.29; corrected P<.001) and ≥60 years (adjusted β coefficient=0.25, 95% CI 0.1-0.38; corrected P<.001) were determinants of a higher score on isolation acceptability or possibility compared to the age of 20-49 years considered as reference. Finally, the knowledge score regarding SARS-CoV-2 transmission was significantly associated with a lower risk of RT-PCR (reverse transcriptase–polymerase chain reaction) positivity (adjusted odds ratio 0.80, 95% CI 0.69-0.94; corrected P<.03), implying that a 1-point increase in the knowledge score lowers the risk of positivity by 20% on average. Conclusions This study identified factors associated with health literacy regarding SARS-CoV-2 infection in asymptomatic individuals in a large French city’s population. We can confirm that in the context of the COVID-19 pandemic, the determinants of better health literacy are not the same as those in other contexts. It seems critical to obtain a more detailed understanding of the determinants of individual citizens’ behavior, as part of a strategy to combat the large-scale spread of the virus. The harsh experience of this pandemic should teach us how to nurture research to structure customized interventions to encourage the adoption of ad hoc behaviors to engage citizens in adapting behaviors more favorable to their health.