Introduction Prescribers have a major role in preventing antimicrobial resistance (AMR) through appropriate prescribing. However, in countries like Sri Lanka, where continuous professional development is not mandatory for license renewal and antimicrobial stewardship is not implemented, prescribing practices go largely unchecked. Objectives To identify the knowledge on antibiotic use and practices related to antibiotic prescribing among Sri Lankan doctors. Methods This cross-sectional study was conducted in 2020. We used a validated, pretested Google-form based questionnaire with multiple choices, single best answer questions, polar questions (Yes/No) and five-point Likert scale questions. The Google-sheet generated was used for data analysis. Knowledge and practice scores were calculated. Results Of the 262 respondents, 40.1% were males. Majority (61.8%) were aged 25–35-years and in medical practice for 0–5 years (48.9%) while 46.2% had or were engaged in post graduate studies. Knowledge scores ranged from 98.31% to 46.55% [mean:71.27% (SD±10.83); median:71.18% (IQR 64.4–79.7)]. Most (98.09%) obtained ≥50 marks while 45.8% scored more than the mean. The practice scores ranged from 100% to 0% [mean:65.33% (SD±18.16), median:66.67% (IQR53.3–80)]. The majority (81.3%) scored ≥50 in the practice score while 52.3% achieved more than the mean practice score. The knowledge score and the practice score differed significantly (p<0.001, related sample Wilcoxon Signed Rank Test) but the knowledge and practice scores were significantly correlated [Spearman correlation, p<0.001, r = 0.343 (Bias corrected 95% CI 0.237–0.448)]. Knowledge scores and the practice scores were significantly higher in those with or undergoing postgraduate training. Conclusions While the knowledge and practice scores were high, and knowledge and practice scores were correlated, the practices score was lower than that of knowledge indicating the need to encourage correct practices through means other than solely promoting knowledge.