The Gram-positive bacteria S. pneumoniae and E. faecalis are a leading cause of hospital- and community-acquired infections. Both bacteria have medical relevance due to the increasing prevalence of strains resistant to multiple antibiotics. As a consequence, the discovery of novel targets and drugs to fight pneumococcal and enterococcal infections is critical. Global transcriptional regulators that respond to specific environmental signals are crucial in the adaptation of pathogenic bacteria to different niches of their eukaryotic hosts. This is likely the case of the MgaSpn and MafR proteins from S. pneumoniae and E. faecalis, respectively. Our research focuses on the molecular characterization of both regulators and their potential paralogues, as well as on the identification of their target genes. Our approach involves a combination of transcriptomics, molecular genetics and biochemistry. By carrying out this research we are providing insights into the links between gene regulation, bacterial adaptation to new environments and virulence processes.