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A key step in the process of isolating microbial natural products is the preparation of an extract from a culture. This step determines which molecules will be available for detection in the subsequent chemical and biological analysis of a biodiscovery pipeline. In the present study we wanted to document potential differences in performance between liquid–liquid extraction using ethyl acetate and liquid–solid extraction using a poly-benzyl-resin. For the comparison of the two extraction protocols, we spiked a culture of Flavobacterium sp. with a diverse selection of natural products of microbial and plant origin to investigate whether the methods were comparable with respect to selectivity. We also investigated the efficiency of the two extraction methods quantitatively, using water spiked with a selection of natural products, and studied the quantitative effect of different pH levels of the aqueous solutions on the extraction yields of the two methods. The same compounds were extracted by the two methods, but the solid-phase extract contained more media components compared with the liquid-phase extract. Quantitatively, the two extraction methods varied in their recovery rates. We conclude that practical aspects could be more important when selecting one of the extraction protocols, as their efficiencies in extracting specific compounds were quite similar.