Rationale: Emergence of worldwide antimicrobial resistance in recent years has been major problem for public health globally. Ensuring patients’ adequate knowledge and proper use of antimicrobials is one of the strategies to control resistance. This study was conducted to evaluate patients’ knowledge and practice regarding antimicrobial use and resistance; as such a study has not been conducted previously in Ethiopia. Cross-sectional study was conducted on 378 patients visiting Felege Hiwot hospital located in the city of Bahir Dar, Northwest Ethiopia, from April 1 to 30, 2014. Data was collected on patients’ knowledge and practice regarding antimicrobial use and resistance using questionnaire, prepared by native language, consisting close ended questions. Results: The participants scored values ranging from 1 to 9 out of 10 with mean value of 4.95 ± 1.88. About 3% of the participants had very good knowledge while 18.8% had good knowledge, 36.0% had average knowledge and 42.3% had poor knowledge about antimicrobial use and resistance. Female patients (AOR=1.838, 95% CI, 1.068, 3.165), those who completed elementary school (AOR=5.286, 95% CI, 1.405-19.883), high school (AOR=20.695, 95% CI, 5.479- 78.161 and higher education (AOR=80.682, 95% CI, 18.670-348.663) were associated with higher knowledge. Among 372 subjects who had ever taken antimicrobials, 56.2% had history of discontinuing antimicrobials before the full dose; 62.6% had ever taken antimicrobials without prescription; and 73.4% had ever missed doses. Conclusion: The findings of the study showed the presence of knowledge gap and malpractice regarding antimicrobial use which will accelerate the rate of development of antimicrobial resistance.