The aim of this study was to investigate the association of 16 microsatellite markers located on chromosome seven with milk production and fertility traits in Egyptian buffalo. Data were collected from 995 lactation records of 296 multiparous Egyptian buffalo during the period from 2000 to 2022. All the studied microsatellite loci were found to be highly polymorphic. The average estimates for molecular genetic parameters across the 16 microsatellite markers were 8.750, 7.306, 0.857, 0.855, 0.741, 2.033, and -0.071 for observed alleles, effective alleles, observed heterozygosity, expected heterozygosity, polymorphic information content, Shannon's information index, and fixation index, respectively. Association analysis revealed significant associations for 14 out of the 16 microsatellite markers with the productive and reproductive traits under investigation (P < 0.05, P < 0.01, and P < 0.001). Specifically, seven microsatellite markers (BM415, BM143, BMC4203, BMS483, RM28, ILSTS93, and ILSTS97) were significantly associated with milk production traits; three markers (BMS2508, BM4311, and BP7) were significantly related to fertility traits; and four markers (BMS2460, BM1329, BM1236, and AFR227) revealed significant associations with multiple traits, encompassing both milk production and fertility traits. The current findings shed more light on unraveling the genetic background underlying milk production and fertility traits in Egyptian buffalo. The significant associations between the investigated microsatellite markers and various phenotypic traits provide evidence that chromosome seven harbors putative causal genetic variants and key candidate genes affecting these economically important traits in Egyptian buffalo.