Several studies have shown that pyrolysis conditions and feedstocks are the key factors influencing biochar chemical and physical properties. The information on the nature of biochar is quite important, especially when this carbonaceous material is intended to be used as a potential soil amendment. In this study, we investigated the formation and characterisation of biochars produced from vacuum pyrolysis of sunflower seed shells (SSS), peanut shells (PS) and Spirulina algae (Sp) at 280 °C (for SSS, PS and Sp) and 350 °C (for PS). As a proxy to test the potential of each biochar as soil amendment, we assessed the germination and growth effects of the biochar water-extractable substances (BWES) at different concentrations (10; 7.5; 5; and 2.5% w/v) on Lactuca sativa. Results showed that the biochar from pyrolysis of PS at 280 °C would be the most suitable soil amendment, since its BWES did not affect germination and exhibited a remarkable growth-promoting effect (50–100%) on roots and stems of L. sativa. In contrast, BWES from SSS, Sp and certain concentrations of PS produced at 350 °C inhibited growth of Lactuca sativa, and particularly BWES of Spirulina dramatically reduced germination, posing a risk for direct application as soil amendment. The presence of carbonyl derivatives in the BWES from PS may be linked to the stimulatory effects of this extract. Aromatics could be responsible for the germination and growth inhibition in the BWES of SSS, while nitrogen organic compounds would enhance the inhibitory effect in BWES from Sp.