The global increase in antibiotic use in food animals has raised awareness of the need to implement interventions to reduce antibiotic misuse. In Peru, small-scale farmers often have limited knowledge about the proper use of antibiotics and tend to use them incorrectly. Bottom-up interventions and participatory approaches can promote sustainable and long-lasting behavioral changes by valuing farmers’ expertise and increasing program acceptability. We aimed to evaluate if participatory interventions can reduce antibiotic misuse on small- and medium-scale laying hen farms from the Ica and Lima regions in Peru. Considering previous surveys and interviews with farmers from the same area, and similar published evidence, the intervention was designed in collaboration with farmers through a prioritization workshop. Prioritized practices included acidification of feed or water, disposal of animal feces, hand hygiene, facility disinfection, and the use of exclusive farm clothing and boots. Intervention farms received training, veterinary advice and a kit consisting of infographics, logbooks, and a container for disposing of antibiotic packaging. Egg production, feed consumption, health incidents and antibiotic use were recorded over a six-month period. A total of 27 intervention farms and 24 control farms were enrolled. During the monitoring period, four farms withdrew from the intervention group and five withdrew from the control group. At baseline, the use of antibiotics as growth promoters was reported by 86.9% (20/23) of intervention farms and 73.7% (14/19) of control farms. Over the intervention period, most practices improved among intervention farms, except for exclusive clothing and boots, while control farms remained largely unchanged. By the end of the study, antibiotic use as a growth promoter remained high (91.3% intervention, 84.2% control). Additionally, other antibiotic misuse—including prophylaxis, metaphylaxis, and treatment of non-bacterial episodes—remained similar between groups (69.6% intervention, 63.2% control), suggesting that participatory interventions alone were insufficient to reduce antibiotic misuse. Quantitative antibiotic use will be estimated using weight-based indicators.