This study evaluates the effect on the 'Splendor' and 'Primoris' strawberry cultivars of different dates of inoculation with Glomus intraradices, an arbuscular mycorrhizal (AM) fungus. Additionally, plants were grown in a soilless growing system with or without Bacillus velezensis at the beginning of the experiment. A completely randomized block design (2 biofertilizer treatments × 2 cultivars × 3 inoculation dates) with 2 replications was used. Each replicate consisted of one bag with 10 plants. Fruit weight, fruit quality, growth parameters, and SPAD values in young leaves were monitored from October 2011 to June 2012 in a greenhouse. At the end of the crop cycle, the microbial population of Bacillus spp. and the Glomus intraradices population were determined from the rhizosphere of the plant. Bacillus velezensis and Glomus intraradices were established in the strawberry soilless growing system. The effect of arbuscular mycorrhizae on strawberry fruit quality was more important than that on growth parameters. Biofertilizer with arbuscular mycorrhizal fungi had an inhibitory effect on fruit quality, as indicated by low TSS, pH, and TA values. The combined effect of the biofertilizer and inoculation dates of Glomus intraradices on growth parameters was more significant in the 'Primoris' cultivar than in 'Splendor'. In both cultivars, an increase in SPAD values was observed from week 12 to week 22 after planting. Depending on the cultivar selected, the date of inoculation may significantly affect plant response to AM fungal colonization in a soilless growing system.