Insect microorganisms significantly affect the diet, well-being and behavior of their hosts. However, the microbiota of spiders, which are important natural enemies of pests, is still largely unknown. To gain insight into the bacterial composition of spiders and their possible roles, we collected five different spider species: Pardosa hortensis, Pholcus phalangioides, Steatoda bipunctata, Steatoda triangulosa (Walckenaer, 1802), and Tegenaria domestica (Clerck, 1757). Mass spectrometry was used to identify the microbiota of each species. After identifying the microbes, the resistence to antibiotics was measured and compared their antibacterial activity with essential oils derived from plants. The antibiotics tested for species-specific antibacterial activity were imipenem (IPM, 30 mg), ciprofloxacin (CIP, 30 µg/disc), linezolid (LZD, 30 µg/disc), tobramycin (TOB, 30 µg/disc), tigecycline (TGC, 30 µg/disc), and tetracycline (TE, 30 µg/disc). We also tested the antibacterial activity of essential oils from Cedar atlantica, Illicium verum, and Pelargonium graveolens. The most frequently identified species were Bacillus mycoides from P. phalangioides, Stutzerimonas chloritidismutans from S. bipunctata, Aerococcus viridans and other species from S. triangulosa, and Bacillus mycoides and Enterococcus faecium from T. domestica. Antibiotic resistance was present in one-third of the isolates, with most antibiotic-resistant bacteria found in T. domestica. The essential oils exhibited high antibacterial activity, particularly against Staphylococcus epidermidis, Bacillus pumilus, Priestia megaterium, and Moraxella osloensis.