We used a high-throughput bicistronic mRNA translation screen to identify natural product inhibitors of eukaryotic protein synthesis from two endophytic actinomycetes, Streptomyces sps. YIM56132 (isolated from Carex baccaus) and YIM56141 (isolated from Fagopyrum cumosum) originating from Yunnan, China. Bioassay-guided fractionation of crude extracts from large-scale fermentation of these strains yielded known compounds including cycloheximide (1), iso-cycloheximide (2), A-75943 (3), actiphenol (6), AH-135Y (7), and phenatic acid A (8), as well as two new cycloheximide congeners, secocycloheximides A (4) and B (5). Structural elucidation relied on HR-MS and NMR spectroscopy, with the stereochemistry of 4 inferred from its conversion to 3 via intramolecular Michael addition during chromatography. Biological evaluations showed that 1 and 2 potently inhibited eukaryotic translation and exhibited broad cytotoxicity against cancer cell lines, while 3 displayed selective cytotoxicity against breast cancer cells. Reduction of 6 (a weak translation inhibitor) to its C-8 hydroxyl analog 9 significantly enhanced inhibitory activity, highlighting the critical role of the C-8 oxidation state in modulating translation inhibition. Additionally, we identified Streptomyces sps. YIM56132 and YIM56141 as new endophytic producers of cycloheximide and its congeners. This study not only expands the structural diversity of cycloheximide derivatives but also provides key insights into their structure-activity relationships, offering a scaffold for the development of more potent eukaryotic protein synthesis inhibitors.