Nontuberculous mycobacteria are the most frequent cause of chronic cervical lymphadenitis in childhood. The aim of the study was to evaluate the performance of IL-2, IL-17, and INF-γ in-house enzyme-linked immunospot assays using a Mycobacterium avium lysate, in order to identify a noninvasive diagnostic method of nontuberculous mycobacteria infection. Children with subacute and chronic lymphadenopathies or with a previous diagnosis of nontuberculous mycobacteria lymphadenitis were prospectively enrolled in the study. Sixty children with lymphadenitis were included in our study: 16 with confirmed infection (group 1), 30 probable infected (group 2) and 14 uninfected (group 3). Significantly higher median cytokine values were found in group 1 vs group 2, in group 1 vs group 3, and in group 2 vs group 3 considering IL-2-based enzyme-linked immunospot assay (p = 0.015, p < 0.001, p = 0.004, respectively). INF-γ-based enzyme-linked immunospot assay results were significantly higher in group 2 vs group 3 (p = 0.010). Differences between infected and uninfected children were not significant considering IL-17 assays (p = 0.431). Mycobacterium avium lysate IL-2 and INF-γ-based enzyme-linked immunospot assays seem to be promising noninvasive diagnostic techniques for discriminating children with nontuberculous mycobacteria lymphadenitis and noninfected subjects.