Purpose To assess the non-prescription use of antibiotics and associated factors in Ambo Town, West Shoa, Oromia, Ethiopia. Methods An institutional-based cross-sectional study design supported with the qualitative study was conducted in Ambo Town from February 1 to March 1, 2020. Data were collected using a pretested semi-structured questionnaire and in-depth interview guide questions. Simple random sampling was used to select retail outlets and systematic random sampling to select study participants. The data analysis was done using SPSS and univariate and multivariate binary logistic regression analysis was performed to identify factors associated with non-prescription use of antibiotics. Thematic framework analysis was applied for the qualitative data. Results From the 421 study sample, a total of 399 participants were interviewed with a 94.8% response rate. Among the study participants, 214 (53.6) were males, 228 (57.1%) were married, 191 (47.9%) were orthodox by religion, and 343 (86%) were Oromo by ethnicity. One hundred seventy-two (43.1%; 95% CI: 38.6, 48.1) of the participants had used non-prescribed antibiotics. Being male [AOR=2.21 95% CI: 1.276, 3.835], residing in rural area [AOR=3.659, 95% CI: 1.479, 9.054], holding diploma [AOR=0.120, 95% CI: 0.025, 0.591], and hold BSC degree [AOR=0.050, 95% CI: 0.007, 0.378], and being farmer [AOR=0.034, 95% CI: 0.004, 0.285] showed significant association with the non-prescription use of antibiotics. Conclusion This study concluded that the non-prescription use of antibiotics 172 (43.1%) was relatively high. Being male, residing in a rural area, holding a diploma, BSc degree, and being a farmer were significantly associated with non-prescription use of antibiotics. So, West Shoa Zone regulatory body should actively focus on the prevention of non-prescription use of antibiotics through health communication and public awareness on the demerits of non-prescription use of antibiotics.