Lyme disease, an infection caused by bacteria of the Borrelia burgdorferi sensu lato complex, is one of the most common vector-borne illnesses in Europe. The infection can affect multiple organs and tissues, leading to a wide range of clinical symptoms. Failure to adequately treat Lyme disease can result in long-term consequences that may lead to severe health complications, significantly impacting the patient's quality of life. The aim of this study was to compare the presence of specific IgM and IgG antibodies in patients with Lyme disease, based on serum testing using the ELISA method with whole-cell and recombinant antigens. Additionally, the study investigated whether both the presence and serum levels of the pro-inflammatory cytokine TNF-alpha correlate with the positivity of these antibodies. Sera from patients tested for Lyme disease were examined by indirect sandwich ELISA for the detection of specific IgM and IgG antibodies against bacteria Borrelia burgdorferi sensu lato. Quantification of TNF-α was performed using the Double Antibody-Sandwich ELISA method. The results were statistically evaluated. The preliminary results of this study suggest that the use of whole-cell antigen compared to recombinant antigen may lead to different results in detecting specific IgM and IgG antibodies, which could impact the diagnosis of Lyme disease. The results also indicate that serum TNF-α concentrations tend to be higher in samples with positive and border-line results. In these samples, the mean TNF-α concentration was higher than in those with negative results for IgM and/or IgG antibodies. Elevated serum TNF-alpha concentrations in patients indicate an inflammatory response and the correlation between TNF-α levels and the presence of specific antibodies may provide valuable insight into the dynamics of the immune response. Furthermore, TNF-α levels could potentially imply the stage of Lyme disease, offering additional diagnostic accuracy.