The objective of this study was to evaluate the effect of inoculation of native arbuscular mycorrhizal fungi (AMF) multiplied on farm in the productivity and quality of different cultivars of sugarcane (Saccharum ssp.) in field conditions by three ratoons. The experimental design was of randomized blocks, in factorial scheme 6 × 2: six cultivars (CTC 1, CTC 7, CTC 9, CTC 16, SP89-1115 and RB925345), with and without AMF inoculation, in three ratoons. The AMF species mix, used to compose the inoculant, were collected from the experimental area, multiplied in trap culture, and applied to the sugarcane planting line. To assess the effectiveness of establishing mycorrhizal symbiosis, root mycorrhizal colonization (MC), spore density (SD), and AMF diversity were quantified. Sugarcane productivity and quality were also obtained by quantifying of the vegetative and technological characteristics. With the inoculation and the increase of viable AMF propagules in the soil resulted in higher MC and SD. Acaulospora scrobiculata and Glomus sp. were the species with the highest frequency of occurrence. Sugarcane productivity and quality were also significantly higher in inoculated treatments, with average increases in the three ratoons of 20% in sugarcane productivity and 30% in sugar production. The increase in sugarcane productivity and quality after AMF inoculation is a positive indicator of the effectiveness this biotechnology has over the production of this commodity.