Hyrtios sponge is known to possess alkaloid compounds that may exhibit in vitro activity against Plasmodium falciparum. The aim of this study was therefore to isolate and characterise the antiplasmodial active compounds of Hyrtios sp. Sponges collected from the island of Podang-Podang, South Sulawesi, Indonesia. In addition, the LC-HRMS analysis was performed on the active fractions of methanol and ethyl acetate extract to evaluate their antiplasmodial activity. We also validated the in silico antiplasmodial activity of PfDHFR-TS with molecular docking, pharmacokinetics, drug likeness, toxicity, and molecular dynamics analysis. The molecular docking studies showed that the synthesized extremes would have high binding affinity to PfDHFR-TS, thus confirming their potential as powerful enzyme inhibitors. Moreover, the pharmacokinetic and drug-likeness calculations showed that all compounds met the requirements for sufficient resistance and bioavailability, indicating potential as therapeutic candidates. The results of the toxicity analysis indicated that the compounds had a relatively good safety profile, but some potential adverse reactions in the renal and cardiac vasculature could not be excluded. Molecular dynamics simulations confirmed that the complexes formed between the ligand and the target were stable, and the low RMSD value indicated that the active site interactions were also quite stable. These observations reinforce the notion that the extract from Hyrtios sp. not only shows remarkable antimalarial activity but also exhibits pharmacological properties of a prospective drug candidate, which encourages further work in the development of malaria combination therapy both in clinical assessment and comprehensive mechanism of action investigation.