This study compares three Romanian Hyssopus officinalis species—H. officinalis f. ruber (HOR), H. officinalis f. albus (HOA), and H. officinalis f. cyaneus (HOC)—evaluating their chemical composition and biological activities, specifically protein denaturation, haemolysis inhibition, and antibacterial effects. Chemical profiles were determined using Gas Chromatography–Mass Spectrometry (GC-MS). The species were cultivated at two distinct locations: the Didactic and Experimental Station DESUSVT and the Agricultural Research and Development Station Lovrin (ARDSL). This study investigates the correlation between chemical composition, biological activities, and local climate data at each site. The results show significant variations in chemical profiles, with species and cultivation location influencing the biological activities. H. officinalis f. albus (HOA) exhibited the strongest antimicrobial activity, particularly against Gram-positive bacteria. The molecular docking analysis highlighted key compounds, such as cyclohexene,4-isopropenyl-1-methoxymethoxymethyl and elemol, with binding solid affinities to microbial and inflammatory proteins. This study provides valuable insights into the chemical and biological properties of Hyssopus officinalis, emphasising its potential in combating microbial infections, protein denaturation, and haemolysis inhibition.