Smoothie drinks are currently very popular drinks sold, especially in fast food establishments. However, smoothies are a significant source of microorganisms with a high level of antibiotic resistance. The study aimed to evaluate the microbiological quality of smoothies purchased in Eastern Bohemia. A higher prevalence of mesophilic aerobic bacteria (5.4–7.2 log CFU/mL), yeast (4.4–5.9 log CFU/mL), and coliform bacteria (3.1–6.0 log CFU/mL) was observed in vegetable smoothies, in which even the occurrence of enterococci (1.6-3.3 log CFU/mL) was observed. However, the occurrence of S. aureus, Salmonella spp., and Listeria spp. was not observed in any sample. Nevertheless, a significant occurrence of resistant microbial strains was observed in all samples. The highest resistance level was found in isolates from smoothie drinks with a dominant vegetable content (green-smoothie drinks). Considerable resistance was observed in Gram-negative rods, especially to amoxicillin (82.2%) and amoxicillin with clavulanic acid (55.6%). Among enterococci, only one vancomycin-resistant strain was detected. The vast majority of isolated strains were able to form biofilms at a significant level, which increases the clinical importance of these microorganisms and their resistance. The highest biofilm production was found in Pseudomonas aeruginosa, Kocuria kristinae, and Klebsiella pneumoniae. Overall, significant biofilm production was also noted among isolates of Candida spp.