Compound 4,4′-(1,4-phenylenebis(thiazole-4,2-diyl))bis(1H-pyrazol-5-amine) (5) was used in the preparation of novel pyrazolo[1,5-a]pyrimidines incorporating a thiazole moiety (6, 7b, 8, 9, and 13–18). the reactions were described via the following reactions: acetylacetone, acetanilide, diethyl malonate, ethyl cyanoacetate, 3-(dimethylamino)-1-phenylprop-2-en-1-one, 2-((dimethylamino)methylene)-3-oxopentane-nitrile, 3-(dimethylamino)-1-(thiophen-3-yl)prop-2-en-1-one, 2-((dimethylamino)methylene)-5,5-dimethylcyclo-hexane-1,3-dione, dimethylformamide-dimethylacetal, and with a mixture of p-nitro benzaldehyde/cyclohexanone, respectively. Gram-positive bacteria B. subtilis and B. thuringiensis, as well as Gram-negative bacteria E. coli and P. aeruginosa, were tested for the newly synthesized compounds' in vitro antibacterial activity. Their in vitro antifungal activity against fungus strains B. Fabae and F. oxysporum was also assessed. The compounds 16–18 had the strongest activity against Gram-positive bacteria, with MIC values = 3.125 µg/mL against B. subtilis, which is equipotent to the drug reference Chloramphenicol, and MIC values = 6.25 µg/mL against B. thuringiensis, which is equipotent to the drug reference Cephalothin. Compounds (13–18) were shown to be superimposable to CNB based on their docking results, with the exception of derivative 18. Furthermore, they demonstrated a strong binding mode with DNA gyrase B's active pocket (PDB: 1KZN) by forming several contacts with the enzyme's essential amino acids in a way that was comparable to CNB.