Antimicrobial resistance (AMR) is a global problem compromising the effective treatment of infectious diseases. The World Health Organization (WHO) is encouraging and promoting awareness creation among health workers as one of its strategies to reduce the rate of emergence and transmission of AMR. Available data on the prescribing behavior of healthcare workers (HCWs) in Nigeria remains incomplete. This study was designed to provide an up-to-date estimate of the knowledge, attitude and antibiotic prescribing behavior of HCWs in Nigeria. This is a cross-sectional study. Self-administered questionnaires were distributed to healthcare workers selected from six states, one each from the 6 geopolitical zones in Nigeria. A multi-stage sampling technique was used to reflect the three tiers of healthcare: primary, secondary and tertiary levels. Quantitative data was summarized using descriptive statistics. All data analysis was done using the Statistical package for social sciences version 26.0. Of the 420 questionnaires distributed, 358 (85.2%) responded. The mean year of practice of the respondents was 9.32 ± 7.8 years. About a half (50.3%) agreed that their prescribing behavior could promote antimicrobial resistance. 49.2% had a good knowledge of AMR and physicians had significantly better knowledge than other HCWs (X  = 69.59, P < 0.001). Several participants prescribed antibiotics for common viral infections such as sore throats (75.7%), measles (37.7%), common cold and flu (21.2%). Over 60.3% admitted prescribing antibiotics just to be on the safe side. In general, 70.9% of the respondents frequently or moderately use practice guidelines while 25.7% often apply the delayed antibiotic prescription (DAP) strategy to reduce antimicrobial prescription. This study reveals an overall moderate level of knowledge of AMR and attitude towards minimizing the emergence of antimicrobial resistance though this did not translate significantly to practice. Further efforts must be made in order to improve rational prescription of antimicrobials among HCWs in Nigeria.