Streptococcus agalactiae (group B Streptococcus, GBS) is a pathogen associated with severe diseases in newborn and immunocompromised patients. One of the commonly used approaches for GBS identification is the CAMP test. It represents enhance of hemolysis when co-cultivating GBS with a β-toxin producing strain of S. aureus. In recent years, in addition to false positive results observed in other bacterial species, CAMP-negative GBS isolates have also been reported, questioning the specificity and sensitivity of the test. CAMP-negative phenotype is characterized by a lack of expression or deletion of cfb gene. According to data, the CovR/S regulatory system, β-hemolysin/cytolysin (cylE), and C5a protease (scpB) genes are possibly involved in the expression of a CAMP-factor. In our study 14 strains out of 294 (4.8%) were tested phenotypically negative for CAMP-factor, but positive for cfb gene. Among the CAMP-negative isolates the antibiotic susceptibility testing revealed resistance rates of 71.4%, 42.9%, and 100.0% for macrolides, lincosamides, and tetracyclines, respectively. Multidrug-resistant (MDR) isolates accounted for 42.9%. Detected serotypes were Ia (35.7%), III (21.4%), V (21.4%), and IV (7.1%). Frequencies of the analyzed virulence factors were as follows: cylE (85.7%) and scpB (92.9%). There was no statistical significance regarding antibiotic resistance and the distribution of the examined virulence genes between strains with CAMP-positive and negative phenotypes. The current study indicated that although the CAMP-test serves as an effective screening diagnostic tool, it is crucial to combine it with additional methods to obtain a conclusive microbiological diagnosis of GBS.