A series of novel 1,3,5-trisubstituted pyrazolines derivatives have been synthesized from chalcones and nicotinic acid hydrazide in two steps. In first step, chalcones were prepared by treatment of 4-hydroxy acetophenone with different substituted benzaldehyde by Claisen-Schimidt Condensation. In second step, various pyrazoline derivatives were prepared by reflux reaction of chalcones with nicotinic acid hydrazide in ethanolic solution. Compounds were confirmed by elemental analyses, IR, ¹H NMR and ¹³C NMR spectral data and were evaluated for antimalarial and antibacterial activity. Compounds 5n (IC₅₀ = 0.022 μM for MRC-2 and IC₅₀ = 0.192 μM for RKL-9) displayed better antiplasmodial activity than the chloroquine (CQ) against chloroquine-sensitive (MRC-2) and chloroquine-resistant (RKL-9) P. falciparum strains. The in vitro cytotoxicity study conducted on the human HepG2 cell line (>30 μM) and selectivity index (100–220) indicate that this series presents an interesting selective antiplasmodial profile. Further, in vitro heme crystallization inhibition assay showed compound 5e inhibited formation of β-hematin more efficiently than CQ. In addition, antibacterial and antifungal evaluations were conducted, compounds 5c, 5i and 5j displayed better antibacterial activity against S. aureus, B. subtilis, E. coli and P. aeruginosa than ciproafloxacin. Antifungal activity of compound 5l against A. niger (MIC-3.25 μg/ml) and C. albicans (MIC-6.5 μg/ml) was found to be better than the standard drug fluconazole.