ABSTRACT The demand for the increased productivity of sugarcane crops has required changes in its production chain, such as the use of pre-sprouted seedlings (PSS) of sugarcane into the chain. In this system, the inoculation of beneficial microorganisms, such as plant growth-promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF), can improve seedling development. The objective was to evaluate the beneficial effect of PGPB and AMF inoculation on sugarcane PSS production system. Experiments were carried out in greenhouse using a commercial substrate with different levels of fertilization to evaluate the plant biomass and nutritional status. The bacteria strains are able to produce indole acetic acid and to amplify the nifH gene. The coinoculation of strains IAC-BeCa-095 with AMF (Glomus macrocarpum and Glomus etunicatum) improved the plant shoot biomass (25%) on the fertilized substrate. The strains IAC-BeCa-088 (Burkholderia caribensis), IAC-RBca5 (Pseudomonas sp.) and IAC-RBca10 (Bacillus sp.) without AMF and fertilization improved the shoot biomass by up to 35%. Coinoculation with strain IAC-BeCa-095 (Kosakonia radicincitans) and AMF improved the shoot and root biomass by up to 27 and 75%, respectively, in the conventionally fertilized substrate, demonstrating a synergistic effect of these microorganism consortia. The use of beneficial microorganisms may be a viable practice in the production of PSS sugarcane. Moreover, this study is the first to demonstrate the synergistic effect of endophytic bacteria (K. radicincitans) or rhizobacteria (Bacillus sp.) with AMF and Pseudomonas sp. or B. caribensis, without AMF inoculum on the production of sugarcane PSS to improve plant growth and plant nutrition.