Microorganisms are only effective when adequate conditions for their survival and development are provided. Among the factors that influence its effectiveness includes the type of soil or culture substrate, which works as an energy source reserve. Therefore, a tomato and a melon crop were established in different cycles to assess the effect of the physicochemical properties of organic substrates based on coconut fibre and vermicompost in three proportions, 0:100, 40:60 and 60:40 (% v:v), on the microbial activity in the rhizosphere when the bacteria Azotobacter vinelandii, Bacillus megaterium and Frateuria aurantia were applied. Concentrations of NO3−, H2PO4−, K+ and Ca2+ in the petiole cellular extract (PCE) were quantified at 60, 90 and 120 days after transplantation (DAT) for tomato and 45 and 65 DAT for melon. We analysed dehydrogenase activity (DHA), acid phosphatase activity (FTA) and β-glucosidase activity (β-GLU). In order to maintain optimal volumetric moisture for the survival of microorganisms, automatic control was used to manage the irrigation frequency between 22%–28%. The results showed that physicochemical substrate properties, by incorporating 40% vermicompost into the coconut fibre mixture, increased enzymatic activity. Plants that were inoculated with Azotobacter vinelandii and Frateuria aurantia showed an improvement in NO3− and K+ assimilation achieving highest yields.