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Bovine tuberculosis and paratuberculosis are endemic in many areas worldwide. This work aims to study cytokines production and gene expression profiles of bovine macrophages infected with <i>Mycobacterium bovis</i> and <i>Mycobacterium paratuberculosis</i> subsp. <i>avium</i> (MAP) strains to identify potential diagnostic biomarkers. Bovine bone marrow stem cells were differentiated into macrophages and subsequently infected in vitro with different spoligotypes of <i>M. bovis</i> and MAP field strains (as single infections and coinfections), using different multiplicity of infection. Supernatant and cell pellets were collected 24 h, 48 h, and one week post-infection. Preliminarily, gene expression on cell pellets of IL-1β, IL-2, INFγ, IL-6, IL-10, IL-12, and TNFα was assessed by qRT-PCR one week p.i. Subsequently, IL-1β and IL-6 were measured by ELISA and qRT-PCR to investigated their production retrospectively 24 h and 48 h p.i. A variability in macrophages response related to the concentration of mycobacteria, the coinfection with MAP, and <i>M. bovis</i> spoligotypes was identified. An early and constant IL-6 increase was observed in the <i>M. bovis</i> infection. A lower increase in IL-1β was also detected at the highest concentration of the two <i>M. bovis</i> spoligotypes one week post-infection. IL-6 and IL-1 β production was reduced and differently expressed in the MAP infection. IL-6 appeared to be the earliest cytokines produced by bovine macrophages infected with <i>M. bovis</i>.