The use of biostimulants consisting of plant growth promoting rhizobacteria (PGPR) is rapidly expanding in horticulture in recent years. In the current study, a novel mix of six Bacillus sp. strains was tested as a PGPR biostimulant in two experiments with zucchini squash (Cucurbita pepo L.) cultivated in the greenhouse soil and in an open field, respectively. In both experiments, seeds of the local landrace ‘Kompokolokytho’ and the commercial hybrid ‘ARO-800’ were either inoculated or non-inoculated with the PGPR biostimulant. The application of the six Bacillus sp. strains increased both the vegetative growth and the yield of zucchini squash, and these effects were associated with significantly higher shoot phosphorus levels in both experiments and both genotypes. Furthermore, at the end of the cultivation, the colony forming units of Bacillus sp. were appreciably higher in plants originating from inoculated compared to non-inoculated seeds, indicating that the tested mix of Bacillus sp can be successfully applied through seed inoculation. ‘ARO-800’ produced more vegetative and fruit biomass than ‘kompokolokytho’ under greenhouse cropping conditions, while in the open-field crop both genotypes performed equally, presumably because the local landrace is traditionally grown in open fields, and thus it is not well adapted to greenhouse cropping.