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 Mycobacterium bovis and Mycobacterium paratuberculosis subsp. avium (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 M. bovis 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 M. bovis spoligotypes was identified. An early and constant IL-6 increase was observed in the M. bovis infection. A lower increase in IL-1β was also detected at the highest concentration of the two M. bovis 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 M. bovis.