Abstract Background Freezing human milk for long periods may reduce its antibacterial properties. We assessed antimicrobial effects of human milk against Escherichia coli after 6 months of freezing and to explore daily variations in both fresh and frozen milk to optimize storage protocols. Methods A study analyzed 60 breast milk samples from 30 healthy mothers with infants aged 15–30 days, conducted from February to May 2025. The human milk expressed during the daytime and at nighttime was tested for antimicrobial effects against E coli. Fresh samples were analyzed within 24 h, and frozen samples were stored at −80°C for 6 months. Bacterial growth was assessed by diluting milk with E coli , and statistics were calculated using colony counts on MacConkey agar. Results Fresh human milk had a median E coli count of 6.5, much lower than 225 in frozen samples stored for 6 months ( P < 0.001), showing a 97.1% drop in antimicrobial quality. Daytime frozen samples had lower microbial counts (median 95) than nighttime samples (median 120, P = 0.006). Higher nighttime counts appeared in frozen samples from primiparous women, cesarean deliveries, and male infants. Maternal age correlated positively with daytime frozen milk microbial counts ( r = 0.421, P = 0.021), whereas gestational age correlated negatively with counts in both daytime ( r = −0.583, P < 0.001) and nighttime samples ( r = −0.562, P = 0.001). Conclusion Frozen human milk samples substantially lose antibacterial properties after 6 months, with variations influenced by time of day. Samples from primiparous, breastfeeding, male sex infants and those who had cesarean deliveries show diurnal variations following the cryopreservation process.