Microbial contamination in shopping trolleys (eighty five) by considering different supermarkets (seven) from three major food companies in Las Palmas de Gran Canaria (Spain) was determined. The two sampled areas were trolley handles and food trolley baskets-child seats. Samples were analyzed by selective and differential microbiological culture media. E. coli four (2.4%) indicative of faecal contamination, Klebsiella pneumoniae twelve (6.5%) and Citrobacter freundii, six (5.1%), which have been isolated from human faecal samples, were isolated from trolleys; Pseudomonas rhodesiae, five (4.25%), and Pseudomonas fluorescens, three (2.55%), which both evidenced environmental contamination. Significant differences among the companies were found for the Enterobacteriaceae and coliforms. Regarding location, these differences (p < 0.003) were observed only for the coliform rates, which were higher in trolleys located outside. The results of this study suggest the implementation of cleaning and disinfection programmes to improve trolley sanitation, and to reduce exposure to both potential pathogenic and transmitting bacterial infections.