Three novel borosilicate bioactive glasses (BBGs) were prepared and used to investigate their bioactive and antibacterial properties. The BBGs were prepared by melt quenching using different glass modifiers, i.e. Mg2+, Ca2+ and Sr2+, and their amorphous nature was confirmed by X-ray diffraction. Scanning electron microscopy with energy dispersive X-ray spectroscopy allowed the visualization of apatite-like structures upon 7 days of immersion in simulated body fluid. BBG-Ca generated surface structures with a Ca/P ratio ≈ 1.67, while the surface of the BBG-Sr was populated with structures with a Sr/P ratio ≈ 1.7. Moreover, bacterial tests showed that the BBG-Mg and BBG-Sr glasses (at concentrations of 9, 18, 36 and 72 mg/mL) present antibacterial characteristics. In particular, BBG-Sr, at concentrations of 9 mg/mL, exhibited bacteriostatic activity against Pseudomonas aeruginosa, and at concentrations ≥ 18 mg/mL it was able to eradicate this bacterium. These results evidence an antibacterial activity dependent on the BBGs composition, concentration and bacterial species. Cellular studies showed that the developed BBGs do not present statistically significant cytotoxic effect against Saos-2 cells after 3 days of culture showing better performance (in the cases of BBG-Ca and BBG-Sr) than commercial 45S5 Bioglass up to 7 days of culture. Overall, this study demonstrates that BBGs can be effectively designed to combine bioactivity and intrinsic antibacterial activity targeting bone tissue engineering applications.