Azoxystrobin (AZO) is widely used to prevent and treat fungal diseases in important crops but can also impact non-target organisms, including mammals, amphibians, aquatic, and soil organisms. Collembolans play important roles in ecosystems as decomposers, fungal feeders, and regulators of microbes. This study aimed to investigate the effects of AZO on Collembola Folsomia candida using a reproduction test and assess biomarker responses over different time intervals (3, 5, 7, 14, and 28 days). Results showed AZO negatively affected reproduction at concentrations of 50, 100, and 200 mg./kg, resulting in decreases of 48.3%, 64.5%, and 81.3%, respectively, compared to the control. Adult survival remained unaffected. The estimated EC50 (reproduction) in artificial soil was 61.28 mg kg−1. Biomarker responses varied with concentration and time. Protein and glycogen concentrations increased with exposure time, while lipid content was affected initially but returned to control levels by day 28. Oxidative stress biomarkers (CAT, SOD, GST, TBARS) indicated AZO induced oxidative stress, intensifying over time. After 28 days, MDA concentrations were significantly elevated compared to the control, suggesting the antioxidant system is overwhelmed which caused damage to lipid membranes. This study showed that azoxystrobin caused negative effects at molecular and population level on non-target species of Collembola.