Bioassay-guided isolation based on the antifungal activity of a culture broth of the marine-derived actinomycete Streptomyces caniferus CA-271066 led to the discovery of new 36-membered polyol macrolides, caniferolides A-D (1-4). Their connectivity was determined by spectroscopic methods including ESITOF-MS and 1D/2D NMR. The relative stereochemistry of each stereocluster in these compounds was established using NOE analysis, the universal database method and J-based configuration analysis, further assisted by comparisons with NMR data of structurally related macrolides. Genome sequencing followed by detailed bioinformatics analysis led to the identification of the corresponding biosynthetic gene cluster and allowed the prediction of the stereochemical outcome of their biosynthesis, confirming the relative stereochemistry of each stereocluster already determined by NMR and establishing their stereochemical relationship, ultimately rendering the absolute configuration of all chiral centers. Furthermore, based on our results and already published data, it has been possible to derive the complete absolute configuration of the related macrolides PM100117 and PM100118, astolides A and B, and deplelides A and B. Caniferolides A-D have shown pronounced antifungal activity against Candida albicans and Aspergillus fumigatus alongside antiproliferative activity against five human tumoral cell lines.