Managing plant fertilization is a major concern of greenhouse growers to achieve sustainable production with growing media (GM). Organic fertilization is popular but is more difficult to control, since organic compounds need first to be mineralized by microbes. After 7, 14, 28, and 56 days of incubation, we investigated the response of microbial activities and nutrient releases from three frequently used organic fertilizers (horn and two plant-based fertilizers) in three frequently employed GM types (peat, coir, and bark). We measured pH, electrical conductivity, nutrient contents (NH4+-N, NO3--N, PO43--P, SO42--S), and enzyme activities (beta-1.4-glucosidase, urease, acid phosphatase, arylsulfatase). After fertilization, microbes in coir expressed all the C, N, P, and S functions studied, making related nutrients available. In peat and bark, some C, N, P, and S-related pathways were locked. Peat presented high NH4+-N and PO43--P releases linked to high acid phosphatase and beta-glucosidase activities, while bark showed high nitrification rates but weak enzyme activities. Fertilizer types modulated these responses with lower activities and nutrient releases with horn. Our results contributed to better understanding mineralization processes in GM, showing different microbial responses to fertilization. This study pointed out the necessity to look deeper into microbial functions in GM optimizing biological and physicochemical properties.