The response of different bacterial species to reduced water availability was studied using a simple relative humidity gradient technique. Interestingly, distinct differences in morphology and growth patterns were observed between populations of the same species growing at different relative humidity. Gram-positive cocci increased in cell size as they approached humidity growth limits and staphylococcal species started growing in tetrad/cubical formations instead of their normal grape-like structures. Gram-negative rods displayed wave-like patterns, forming larger waves as they became increasingly filamentous at low humidity. In contrast, cells of the Gram-positive bacterium Bacillus subtilis became shorter, curved, and eventually almost coccoid. Moreover, B. subtilis started to sporulate at low humidity. The altered morphology and/or growth patterns of bacteria growing at low humidity might be more ecologically relevant than their textbook appearance at high humidity since their natural habitats are often dry. Transmission electron microscopic analyses revealed that staphylococci grown at low humidity have significantly thickened cell walls, which may explain why these cells displayed increased resistance to vancomycin. We conclude that our relative humidity gradient technique is widely applicable for investigating effects of relative humidity on microbial survival, growth and competitive success at solid–air interfaces, making it a versatile tool in microbial ecology.