Abstract Background Africa is challenged by the emergence of antimicrobial resistance (AMR). In order to improve patient management and to optimise approaches to curb the spread of antimicrobial resistance, we examined knowledge and perceptions of AMR and antibiotics prescription practices of HCW (healthcare workers) in Lambaréné, Gabon. Methods We conducted a self-administered, questionnaire-based survey in HCW at the regional referral hospital, a medical research centre, and peripheral health care facilities. The proportions of correct responses to questions were determined and compared between physicians and nurses using Fisher’s Exact test. Results A total of 47 HCW took part in the survey. Of those, 64% (30/47) recognised antibiotic resistance as a major public health issue in Gabon, but only 14/47 (30%) recognised it as a problem in their health facility. Of note, 37/47 (79%) recognised excessive use of antibiotics without microbiological confirmation in case of infection, and buying antibiotics without a prescription, as possible cause of antimicrobial resistance. Some HCW (28%; 13/47) reported having prescribed antibiotics because the patients asked for them; and a total of 15/47 (32%) responded that antibiotics could help patients recover faster when added to malaria treatment. Compared to nurses, most of the physicians recognised that excessive use of antibiotics without microbiological confirmation of infection could contribute to AMR spread (18/19 (95%) vs 19/28 (68%); p = 0.028). Conclusion Most HCW recognised AMR as public health issue. However, a quarter of the participants did not know about the causes fostering the emergence of antimicrobial resistance. There is a need to perform regular HCW training in antimicrobial prescription, and to set up an antimicrobial stewardship program.