The search for novel potent anticancer and antimicrobial agents is considered a rapidly advancing field and viewed as a constantly evolving area within medicinal chemistry. In this work, a series of novel spiropyrazoline oxindole scaffolds were synthesized based on domino Knoevenagel–Michael cyclization reactions. These compounds were tested for their in vitro cytotoxic activity against HePG-2 [human hepatocellular carcinoma cell line] and MCF-7 [breast cancer cell line]. Compounds 5a, 5b, and 5d showed potent activity against the HePG-2 cell line. Compound 5a was the most potent one and showed activity against both cell lines, with IC50 (μg mL−1) values of 12.3 and 17.3, respectively, compared to adriamycin, with IC50 (μg mL−1) values of 21.6 and 25.5, respectively. An in silico study, encompassing both molecular docking and MD simulations, highlighted the potential of compounds 5a and 5b as potent therapeutic agents targeting the 4I4T protein, compared to three commercially available drugs, namely, adriamycin, sunitinib, and spirobrassinin. This study demonstrates the importance of spiropyrazoline oxindoles for the development of new and potent cancer treatments. The MD simulations confirmed that compound 5a has a more stable and stronger interaction with the 4I4T protein, making it a promising candidate for further development. An in silico study was conducted to support the experimental results, and another one to show the binding affinity with the PBP2a receptor protein of S. aureus for future research. Compound 4a showed a binding affinity energy of −7.9 kcal mol−1, compared to −6.5 kcal mol−1 for linezolid and −6.3 kcal mol−1 for penicillin.