Two novel antibiotic spiculisporic acid analogues, named as spiculisporic acid F (1) and G (2), and two known compounds, (-)-spiculisporic acid (3) and secospiculisporic acid B (4), were isolated by bioactivity-guided fractionation from the fermentation broth of the sea urchin-derived Aspergillus candidus strain HDf2. Their structures were unambiguously established by comprehensive analysis of 1D and 2D NMR, and high-resolution MS spectra, and by comparison with known compounds. Biological experiments demonstrated that compounds 1 and 2 displayed antibacterial activity against Gram-negative Pseudomonas solanacearum and Gram-positive Staphylococcus aureus, but showed no cytotoxicity against SGC-7901 human gastric adenocarcinoma and SPC-A-1 human lung adenocarcinoma tumor cell lines. This is the first critical evidence identifying spiculisporic acid derivatives as a potential bio-control agent for the soil borne pathogen P. solanacearum (E. F. Smith) Smith. These findings provide further insight into the chemical and biological activity diversity of this class of compounds.